[Objective] The aim of the research was to analyze the resistance of binary insect-resistant transgenic soybean to Heliothis viriplaca.[Method]In this experiment, resistance analysis of the stabilized binary insect-re...[Objective] The aim of the research was to analyze the resistance of binary insect-resistant transgenic soybean to Heliothis viriplaca.[Method]In this experiment, resistance analysis of the stabilized binary insect-resistant transgenic soybean to Heliothis viriplaca was conducted in lab and in field conditions.[Result] The results indicated that the leaves of insect-resistant transgenic soybeans T5-150 and T5-195 showed lighter damage than those of non-transgenic soybeans. Meanwhile, the Heliothis viriplaca larvae fed on leaves of these two transgenic soybeans were characterized by less leaf consumption, shortening survival day, slower development and less pupation.[Conclusion]It was concluded that insect-resistance of transgenic soybean to Heliothis viriplaca was increased dramatically and the research provided a reference for selecting binary insect-resistant transgenic soybean to Heliothis viriplaca.展开更多
[Objective] Taqman Quantitative PCR technique was adopted to detect the copies of exogenous nos terminator in transgenic hybrid soybean.[Method] With soybean Lectin as the endogenous reference gene,and gene complex DN...[Objective] Taqman Quantitative PCR technique was adopted to detect the copies of exogenous nos terminator in transgenic hybrid soybean.[Method] With soybean Lectin as the endogenous reference gene,and gene complex DNA in non-GMO soybeans as the endogenous reference standard,the method of gradient dilution was used for separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies,and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation.[Result] The standard curve equation of endogenous reference gene is y=-3.422x+35.201,R2=0.998;and the standard curve equation of exogenous gene is y=-3.348x+34.890,R2=0.999.Nos terminator and its lower boundary sequences in transgenic soybean is of single copy.[Conclusion] The study has provided a theoretical basis for determining exogenous gene copies in transgenic soybean.展开更多
ObjectiveThe aim was to understand the effects of transgenic DREB soybean on the ammonia-oxidizing bacteria. MethodThe diversity of the cto gene in pot-planted transgenic soybean and near-isogenic non-transgenic soybe...ObjectiveThe aim was to understand the effects of transgenic DREB soybean on the ammonia-oxidizing bacteria. MethodThe diversity of the cto gene in pot-planted transgenic soybean and near-isogenic non-transgenic soybean under normal water condition and drought stress was analyzed by PCR-DGGE and sequence analysis. ResultRhizosphere community diversity of ammonia-oxidizing bacteria showed no difference between the treatments of transgenic soybean and its non-transgenic isolines, moreover transgenic soybean under normal water condition and drought stress improved the diversity of the ammonia-oxidizing bacteria in the harvest time. The phylogenetic analysis revealed that all the sequences of excised DGGE bands were closely related to members of the genus Nitrosovibrio and Nitrosospira of the β-subclass Proteobacteria. ConclusionTransgenic DREB soybean has no adverse impact on soil ammonia-oxidizing bacteria.展开更多
Glycogen synthase kinase 3 (GSK3) is a kind of sedne/threonine kinase widely found in eukaryotes. Many plant GSK3 kinases play important roles in regulating stress responses. This study investigated BRASSINOSTEROID-...Glycogen synthase kinase 3 (GSK3) is a kind of sedne/threonine kinase widely found in eukaryotes. Many plant GSK3 kinases play important roles in regulating stress responses. This study investigated BRASSINOSTEROID-INSENSITIVE 2 (GmBIN2) gene, a member of the GSK3 protein kinase family in soybean and an orthologue of Arabidopsis BIN2/AtSK21. GmBIN2 expression was increased by salt and drought stresses, but was not significantly affected by the ABA treatment. To examine the function of GrnBIN2, transgenic Arabidopsis and transgenic soybean hairy roots were generated. Overexpression of GmBIN2 in Arabidopsis resulted in increased germination rate and root length compared with wild-type plants under salt and mannitol treatments. Overexpression of GmBIN2 increased cellular Ca2~ content and reduced Na~ content, enhancing salt tolerance in transgenic Arabidopsis plants. In the soybean hairy root assay, overexpression of GmBIN2 in transgenic roots also showed significantly higher relative root growth rate than the control when subjected to salt and mannitol treatments. Measurement of physiological indicators, including proline content, superoxide dismutase (SOD) activity, and relative electrical conductivity, supported this conclusion. Furthermore, we also found that GmBIN2 could up-regulate the expression of some stress-related genes in transgenic Arabidopsis and soybean hairy roots. Overall, these results indicated that GmBIN2 improved tolerance to salt and drought in transgenic Arabidopsis and soybean hairy roots.展开更多
A fast and efficient recognition method of transgenic lines will greatly improve detection efficiency and reduce cost.In this study,we successfully identified the transgenic soybean plants by the color.We isolated a G...A fast and efficient recognition method of transgenic lines will greatly improve detection efficiency and reduce cost.In this study,we successfully identified the transgenic soybean plants by the color.We isolated a GmW1 gene encoding a flavonoid 3'5'-hydroxylase from a soybean cultivar ZH42(purple flower).We found that purple flowers occurred in the overexpression lines in the Jack and Williams 82 backgrounds(white flower).All plants with purple flowers were positive,and this trait seems stably inherited in the offspring.We have also obtained the editing plants,which were classified into three types according to the different flower colors appeared.We analyzed the phenotype and the homozygous types of the T_1mutants.We also found that a correspondence between flower color and stem color.This study provides a visible color reporter on soybean transformation.It can be quickly and early to identify the transgenic soybean plants by stem color of seedlings,which substantially reduces the amount of labor and cost.展开更多
TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybean...TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x+35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x+35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.展开更多
Glyphosate-tolerant soybean is the most widely planted genetically modified crop worldwide. However, soybean remains recalcitrant to routine transformation because of the low infection efficiency of Agrobacterium to s...Glyphosate-tolerant soybean is the most widely planted genetically modified crop worldwide. However, soybean remains recalcitrant to routine transformation because of the low infection efficiency of Agrobacterium to soybean and lack of useful selectable markers. In this study, several Agrobacterium strains and cell densities were compared by transient expression of the GUS gene. The results showed that Agrobacterium strain Ag10 at cell densities of OD_(600) of 0.6–0.9 yielded the highest infection efficiency in Agrobacterium-mediated soybean cotyledonary node transformation system. Meanwhile, a simple and rapid method was developed for identification of glyphosate tolerance in putative T_0 transgenic plants, consisting of spotting plantlets with 1 μL Roundup~?. The whole cycle of genetic transformation could be shortened to about 3 mon by highly efficient selection with glyphosate during the transformation process and application of the spot assay in putative T_0 transgenic plantlets. The transformation frequency ranged from 2.9 to 5.6%. This study provides an improved protocol for development and identification of glyphosate-tolerant transgenic soybeans.展开更多
The introduction of genetically modified(GM) soybean into farming systems raises great concern that transgenes from GM soybean may flow to endemic wild soybean via pollen. This may increase the weediness of transgenic...The introduction of genetically modified(GM) soybean into farming systems raises great concern that transgenes from GM soybean may flow to endemic wild soybean via pollen. This may increase the weediness of transgenic soybean by increasing the fitness of hybrids under certain conditions and threaten the genetic diversity of wild soybean populations. Although pollen-mediated gene flow between GM crops and wild relatives is dependent on many factors, the sexual compatibility(SC)determined by their genetic backgrounds is the conclusive factor. The considerable genetic variation among wild soybean populations may cause compatibility differences between different wild and cultivated soybeans. Thus, an evaluation of the SC between transgenic soybean and different wild soybeans is essential for assessing the environmental consequences of cultivated soybean–wild soybean transgene flow. The podding and seed sets were assessed after artificial hybridization using transgenic glyphosate-resistant soybean as the paternal parent and 18 wild soybean populations as the maternal parents. Then, the average number of filled seeds produced in 200 flowers(AFS) was calculated for each wild soybean under natural self-pollination as well as under artificial crossing with transgenic soybean. Finally, the index of cross-SC was calculated(ICSC) as the ratio of the AFS of wild soybean artificially crossed with transgenic soybean and the AFS of naturally self-pollinated wild soybean. The results demonstrated that after self-pollination and crossing with transgenic soybean, the average podding rates of 18 wild soybean populations ranged within 96.50–99.50% and 4.92–18.03%, and the average filled seed numbers per pod varied from 1.70 to 2.69 and 0.20 to 0.48, respectively. The results showed that approximately 89% of wild soybeans displayed either medium or higher than medium SC with transgenic soybean(ICSC>1.0%). This implied the high possibility of gene flow via pollen from transgenic soybean to wild soybean.展开更多
Dongnong50 was overexpressing TaDREB3, a stress-tolerance gene, and transgenic progenies with TaDREB3 showing stable heredity characters were obtained in this study. The seeds of T4 generation were sowed on saline lan...Dongnong50 was overexpressing TaDREB3, a stress-tolerance gene, and transgenic progenies with TaDREB3 showing stable heredity characters were obtained in this study. The seeds of T4 generation were sowed on saline land of which salt concentration was 0.19% and pH was 8.43, and agronomic traits including growth period, plant height, numbers of main nodes, branching numbers, pods per plant, numbers of seeds per plant, and weight of 100-seed were collected and comparing to control plants Dongnong50 were made. The impacts of TaDREB3 gene on agronomic traits of soybeans under high concentration of salt were discussed. The results showed that the growth period was not altered in transgenic line with TaDREB3 under high concentration of saline and alkali, the extremely significant or significant increase in the numbers of main nodes, pods per plant and in branching numbers and other agronomic traits in transgenic lines in comparing with non-transgenic lines was an indicator of their resistance to saline-alkali stress展开更多
Transgenic food safety is a high-profile public health issue in worldwide, especially transgenic soybean (Glycine max L.) oil. To rapidly and effectively detect transgenic components of soybean oil, in the present stu...Transgenic food safety is a high-profile public health issue in worldwide, especially transgenic soybean (Glycine max L.) oil. To rapidly and effectively detect transgenic components of soybean oil, in the present study, we isolated DNA from transgenic soybean oil by modified method, and employed the multiplex PCR method to identify targeted genes, including CaMV35S promoter, Nos terminator, NPTII, CP4-EPSPS and endogenous gene Lectin. The research aims to build a method which is accurate, rapid and reliable for detection of genetically modified soybeans oil. The targeted gene including DNA was successfully established by the improved method, and then amplified by PCR. Five genes are simultaneously specifically detected. Commercial soybean, genetically modified soy bean and oil were detected with the Multiplex PCR. The improved method of DNA extraction was rapid and accurate to extract high quality total DNA which was amplified by PCR. The method could eliminate the PCR inhibitor. A way of detecting the genetically modified soybean and Oil was set up in this study.展开更多
A PCR-ELISA method for detecting the glyphosate resistant transgenic soybean was established and optimized. The results showed that the key parameters of PCR-ELISA were as follows: the concentration of digoxin tag pr...A PCR-ELISA method for detecting the glyphosate resistant transgenic soybean was established and optimized. The results showed that the key parameters of PCR-ELISA were as follows: the concentration of digoxin tag probe was 0.5 μmol · L^-1, the time of hybridization reaction was 15 min and the chromogenic reaction should last for 30 min. The sensitivity and the repeatability of our PCR-ELISA method were evaluated, and the results showed that it could be detected when the concentration of DNA template from transgenic soybean samples was 0.01% or higher, and the coefficient of variation of this method was less than 5% in our research condition. These results suggested that PCR-ELISA method establishment in this study had good repeatability and high precision for detecting the transgenic soybean samples.展开更多
ENHANCER OF SHOOT REGENERATION(ESR1)is an important regulator of plant regeneration in vitro,which promotes regeneration of plant.In this study,transgenic positive plants with normal expression of proteins were screen...ENHANCER OF SHOOT REGENERATION(ESR1)is an important regulator of plant regeneration in vitro,which promotes regeneration of plant.In this study,transgenic positive plants with normal expression of proteins were screened by molecular assay.Through the study of the transgenic plants and the control Dongnong 50,the difference between immature embryo-induced callus and induced shoot bud was observed.The increase in callus weight indicated that GmESR1 gene accelerated the formations of shoot buds.By measuring the changes of hormone in the process of induction callus of transgenic plants,it was found that the contents of indole-3-acetic acid(IAA)and zeatin(ZT)in transgenic lines were significantly increased.It could be concluded that GmESR1 gene promoted the accumulation of hormone and affected regeneration process.In addition,this study also verified the interaction between GmBIM1 gene and GmESR1 gene by bimolecular fluorescence complementation(BiFC).展开更多
Drought is a bottleneck for worldwide soybean production which is getting more serious as the climate continues to worsen. Dehydration responsive element binding(DREB) is a kind of transcription factor that regulate...Drought is a bottleneck for worldwide soybean production which is getting more serious as the climate continues to worsen. Dehydration responsive element binding(DREB) is a kind of transcription factor that regulates the expression of stress tolerance-related genes in response to drought, high salinity and cold stress in plant. Soybean with DREB gene possesses the drought resisting capability which is helpful to increase the yield. However, the potential risk of genetically modified plants(GMPs) on soil microbial community is still in debate. In order to understand the effects of transgenic DREB soybean on the nitrogen-fixing bacteria, the diversity of nif H gene in pot experiments planted transgenic soybean and near-isogenic nontransgenic soybean under normal water condition and drought stress condition was analyzed by PCR-DGGE and sequence analysis. The results showed that transgenic soybean under normal water condition decrease the diversity of the nitrogen-fixing bacteria in the seeding stage and flowering stage, but had no notable effect in other stages. Under drought stress, transgenic soybean reduced the diversity of the nitrogen-fixing bacteria in the flowering stage, but had no notable effects on other stages. Phylogenic analysis revealed that g7, g13, g15 and g19 had a close relationship with Alphaproteobacteria, g12 had a close relationship with Azonexus, others were related to Betaproteobacteria and Burkholderia.展开更多
[Objectives] This study was conducted to investigate the possibility of screening for herbicide resistance in the early stage of rapes growth. [Methods] Eight treatments were set for a herbicide concentration experime...[Objectives] This study was conducted to investigate the possibility of screening for herbicide resistance in the early stage of rapes growth. [Methods] Eight treatments were set for a herbicide concentration experiment. [Results] Rape sprouts were highly sensitive to the herbicide glyphosate, and even a very low concentration of glyphosate solution(187 mg/L) was sufficient to kill most of the rape seedlings, while the genetically modified herbicide-tolerant rape could tolerate higher concentrations of the herbicide. Low concentrations of glyphosate could be used for screening at the sprout stage, and the results of herbicide-tolerance screening were consistent with PCR testing. [Conclusions] This study lays a technical and material foundation for accelerating the cultivation of new herbicide-resistant rape varieties suitable for rapeseed production areas in the lower reaches of the Yangtze River.展开更多
Genetically modified(GM) organisms are widely adopted. However, their safety assessments and control are still of special concern to the public. Identifying and localizing transgene insertion is an essentially prerequ...Genetically modified(GM) organisms are widely adopted. However, their safety assessments and control are still of special concern to the public. Identifying and localizing transgene insertion is an essentially prerequisite step. In this study, 2 independent transgene soybean lines were selected(LB4-AtDCGS-1-20-5-2 and CGS-ZG11) as typical cases. Both lines contained expression cassette of At-DCGS that encoding a feedback-insensitive cystathionine gamma-synthase to produce higher level methionine(Met). LB4-AtDCGS-1-20-5-2 was whole genome sequenced with one paired-end 500 bp library and two mate-paired 1 kb and 2 kb libraries using Illumina HiSeq sequencing platform. CGS-ZG11 was sequenced with only one paired-end 500 bp library. Both genomes were assembled,and 2 scaffold sequences(1 for each line) were screened out by aligning with transgene.Then the transgene insertion and its flanking regions in soybean genome were further identified and confirmed by PCR cloning and Sanger sequencing. Results showed that these 2 transgene lines had single copy of inserted transgene. Their transgene insertion contents were identified, which facilitates further safety assessment. These results indicated that genome assembly using high throughput sequencing is a powerful tool for identifying transgene insertions, even with limited knowledge.展开更多
[Objective] The aim was to establish an event-specific qualitative PCR method for transgenic soybean MON89788.[Method] Firstly,the 3′-junction sequence between host plant DNA and integrated DNA of transgenic MON89788...[Objective] The aim was to establish an event-specific qualitative PCR method for transgenic soybean MON89788.[Method] Firstly,the 3′-junction sequence between host plant DNA and integrated DNA of transgenic MON89788 soybean was isolated using thermal asymmetric interlaced-PCR (TAIL-PCR),and the specific PCR primers were designed based on the 3′-junction sequence.Secondly,the specificity and sensitivity of the qualitative PCR detection methods employing these primers were tested.[Result] 1 142-bp 3′-junction sequence was obtained.According to the sequence,event-specific qualitative PCR method was established,amplifying a 170-bp product specifically from MON89788 event,and the limit of detection was 0.05%,approximately 40 initial template copies.[Conclusion] The method was highly specific,sensitive,and suitable for detection of MON89788 event.展开更多
Glyphosate is a highly efficient, broad-spectrum nonspecific herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)-mediated pathway of shikimic acid. The screening of glyphosate-resistant EPSP...Glyphosate is a highly efficient, broad-spectrum nonspecific herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)-mediated pathway of shikimic acid. The screening of glyphosate-resistant EPSPS gene is a major means for the development of new genetically modified glyphosate-resistant transgenic crop. Currently, the main commercialized glyphosate-resistant soybean contains glyphosate-resistant gene CP4-EPSPS. In this study, a G10-EPSPS gene was reported providing glyphosate resistance in Zhongdou 32. Here, G10-EPSPS gene was introduced into soybeans through Agrobacterium-mediated soybean cotyledon node. PCR, Southern blotting, semi-quantitative RT-PCR, qRT-PCR, and Western blotting were used, and the results revealed that G10-EPSPS had been integrated into the soybean genome and could be expressed steadily at both mRNA and protein levels. In addition, glyphosate resistance analysis showed that the growth of transgenic soybean had not been affected by concentrations of 900 and 2 700 g a.e. ha–1 of glyphosate. All the results indicated that G10-EPSPS could provide high glyphosate resistance in soybeans and be applied in production of glyphosate-resistant soybean.展开更多
文摘[Objective] The aim of the research was to analyze the resistance of binary insect-resistant transgenic soybean to Heliothis viriplaca.[Method]In this experiment, resistance analysis of the stabilized binary insect-resistant transgenic soybean to Heliothis viriplaca was conducted in lab and in field conditions.[Result] The results indicated that the leaves of insect-resistant transgenic soybeans T5-150 and T5-195 showed lighter damage than those of non-transgenic soybeans. Meanwhile, the Heliothis viriplaca larvae fed on leaves of these two transgenic soybeans were characterized by less leaf consumption, shortening survival day, slower development and less pupation.[Conclusion]It was concluded that insect-resistance of transgenic soybean to Heliothis viriplaca was increased dramatically and the research provided a reference for selecting binary insect-resistant transgenic soybean to Heliothis viriplaca.
基金Funded by Program of Technology Bureau of Harbin(2010RFQXN101)Sub-project of Transgenic Significant Specific Project(2008ZX08012-001)~~
文摘[Objective] Taqman Quantitative PCR technique was adopted to detect the copies of exogenous nos terminator in transgenic hybrid soybean.[Method] With soybean Lectin as the endogenous reference gene,and gene complex DNA in non-GMO soybeans as the endogenous reference standard,the method of gradient dilution was used for separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies,and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation.[Result] The standard curve equation of endogenous reference gene is y=-3.422x+35.201,R2=0.998;and the standard curve equation of exogenous gene is y=-3.348x+34.890,R2=0.999.Nos terminator and its lower boundary sequences in transgenic soybean is of single copy.[Conclusion] The study has provided a theoretical basis for determining exogenous gene copies in transgenic soybean.
基金Supported by the Special Scientific Fund for Non-profit Environmental Industry(2010467038)~~
文摘ObjectiveThe aim was to understand the effects of transgenic DREB soybean on the ammonia-oxidizing bacteria. MethodThe diversity of the cto gene in pot-planted transgenic soybean and near-isogenic non-transgenic soybean under normal water condition and drought stress was analyzed by PCR-DGGE and sequence analysis. ResultRhizosphere community diversity of ammonia-oxidizing bacteria showed no difference between the treatments of transgenic soybean and its non-transgenic isolines, moreover transgenic soybean under normal water condition and drought stress improved the diversity of the ammonia-oxidizing bacteria in the harvest time. The phylogenetic analysis revealed that all the sequences of excised DGGE bands were closely related to members of the genus Nitrosovibrio and Nitrosospira of the β-subclass Proteobacteria. ConclusionTransgenic DREB soybean has no adverse impact on soil ammonia-oxidizing bacteria.
基金supported by the funding from the Creative Research Groups of Heilongjiang Province of China(JC2016004)the National Key R&D Program of China(2016YFD0100201-21)+1 种基金the Project of Outstanding Academic Leaders in Harbin,China(2015RQXXJ018)the China Agriculture Collaborative Creation Research System of Miscellaneous Grain Crops
文摘Glycogen synthase kinase 3 (GSK3) is a kind of sedne/threonine kinase widely found in eukaryotes. Many plant GSK3 kinases play important roles in regulating stress responses. This study investigated BRASSINOSTEROID-INSENSITIVE 2 (GmBIN2) gene, a member of the GSK3 protein kinase family in soybean and an orthologue of Arabidopsis BIN2/AtSK21. GmBIN2 expression was increased by salt and drought stresses, but was not significantly affected by the ABA treatment. To examine the function of GrnBIN2, transgenic Arabidopsis and transgenic soybean hairy roots were generated. Overexpression of GmBIN2 in Arabidopsis resulted in increased germination rate and root length compared with wild-type plants under salt and mannitol treatments. Overexpression of GmBIN2 increased cellular Ca2~ content and reduced Na~ content, enhancing salt tolerance in transgenic Arabidopsis plants. In the soybean hairy root assay, overexpression of GmBIN2 in transgenic roots also showed significantly higher relative root growth rate than the control when subjected to salt and mannitol treatments. Measurement of physiological indicators, including proline content, superoxide dismutase (SOD) activity, and relative electrical conductivity, supported this conclusion. Furthermore, we also found that GmBIN2 could up-regulate the expression of some stress-related genes in transgenic Arabidopsis and soybean hairy roots. Overall, these results indicated that GmBIN2 improved tolerance to salt and drought in transgenic Arabidopsis and soybean hairy roots.
基金supported by the Agricultural Science and Technology Innovation Program of Chinese Academy of Agriculture Sciences(S2022ZD03)。
文摘A fast and efficient recognition method of transgenic lines will greatly improve detection efficiency and reduce cost.In this study,we successfully identified the transgenic soybean plants by the color.We isolated a GmW1 gene encoding a flavonoid 3'5'-hydroxylase from a soybean cultivar ZH42(purple flower).We found that purple flowers occurred in the overexpression lines in the Jack and Williams 82 backgrounds(white flower).All plants with purple flowers were positive,and this trait seems stably inherited in the offspring.We have also obtained the editing plants,which were classified into three types according to the different flower colors appeared.We analyzed the phenotype and the homozygous types of the T_1mutants.We also found that a correspondence between flower color and stem color.This study provides a visible color reporter on soybean transformation.It can be quickly and early to identify the transgenic soybean plants by stem color of seedlings,which substantially reduces the amount of labor and cost.
基金Supported by the Program of Technology Bureau of Harbin (2010RFQXN101)the Subproject of Transgenic Significant Specific Project (20112X08004-002-002-004)
文摘TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x+35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x+35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.
基金supported by the National Natural Science Foundation of China(31601326)the National Transgenic Major Program of China(2016ZX08004001 and 2016ZX08011003)the China Postdoctoral Science Foundation(2016M591300)。
文摘Glyphosate-tolerant soybean is the most widely planted genetically modified crop worldwide. However, soybean remains recalcitrant to routine transformation because of the low infection efficiency of Agrobacterium to soybean and lack of useful selectable markers. In this study, several Agrobacterium strains and cell densities were compared by transient expression of the GUS gene. The results showed that Agrobacterium strain Ag10 at cell densities of OD_(600) of 0.6–0.9 yielded the highest infection efficiency in Agrobacterium-mediated soybean cotyledonary node transformation system. Meanwhile, a simple and rapid method was developed for identification of glyphosate tolerance in putative T_0 transgenic plants, consisting of spotting plantlets with 1 μL Roundup~?. The whole cycle of genetic transformation could be shortened to about 3 mon by highly efficient selection with glyphosate during the transformation process and application of the spot assay in putative T_0 transgenic plantlets. The transformation frequency ranged from 2.9 to 5.6%. This study provides an improved protocol for development and identification of glyphosate-tolerant transgenic soybeans.
基金financially supported by the National Special Transgenic Project of China(2016ZX08012005)。
文摘The introduction of genetically modified(GM) soybean into farming systems raises great concern that transgenes from GM soybean may flow to endemic wild soybean via pollen. This may increase the weediness of transgenic soybean by increasing the fitness of hybrids under certain conditions and threaten the genetic diversity of wild soybean populations. Although pollen-mediated gene flow between GM crops and wild relatives is dependent on many factors, the sexual compatibility(SC)determined by their genetic backgrounds is the conclusive factor. The considerable genetic variation among wild soybean populations may cause compatibility differences between different wild and cultivated soybeans. Thus, an evaluation of the SC between transgenic soybean and different wild soybeans is essential for assessing the environmental consequences of cultivated soybean–wild soybean transgene flow. The podding and seed sets were assessed after artificial hybridization using transgenic glyphosate-resistant soybean as the paternal parent and 18 wild soybean populations as the maternal parents. Then, the average number of filled seeds produced in 200 flowers(AFS) was calculated for each wild soybean under natural self-pollination as well as under artificial crossing with transgenic soybean. Finally, the index of cross-SC was calculated(ICSC) as the ratio of the AFS of wild soybean artificially crossed with transgenic soybean and the AFS of naturally self-pollinated wild soybean. The results demonstrated that after self-pollination and crossing with transgenic soybean, the average podding rates of 18 wild soybean populations ranged within 96.50–99.50% and 4.92–18.03%, and the average filled seed numbers per pod varied from 1.70 to 2.69 and 0.20 to 0.48, respectively. The results showed that approximately 89% of wild soybeans displayed either medium or higher than medium SC with transgenic soybean(ICSC>1.0%). This implied the high possibility of gene flow via pollen from transgenic soybean to wild soybean.
基金Supported by Project of Education Department of Heilongjiang Province (11551048)
文摘Dongnong50 was overexpressing TaDREB3, a stress-tolerance gene, and transgenic progenies with TaDREB3 showing stable heredity characters were obtained in this study. The seeds of T4 generation were sowed on saline land of which salt concentration was 0.19% and pH was 8.43, and agronomic traits including growth period, plant height, numbers of main nodes, branching numbers, pods per plant, numbers of seeds per plant, and weight of 100-seed were collected and comparing to control plants Dongnong50 were made. The impacts of TaDREB3 gene on agronomic traits of soybeans under high concentration of salt were discussed. The results showed that the growth period was not altered in transgenic line with TaDREB3 under high concentration of saline and alkali, the extremely significant or significant increase in the numbers of main nodes, pods per plant and in branching numbers and other agronomic traits in transgenic lines in comparing with non-transgenic lines was an indicator of their resistance to saline-alkali stress
文摘Transgenic food safety is a high-profile public health issue in worldwide, especially transgenic soybean (Glycine max L.) oil. To rapidly and effectively detect transgenic components of soybean oil, in the present study, we isolated DNA from transgenic soybean oil by modified method, and employed the multiplex PCR method to identify targeted genes, including CaMV35S promoter, Nos terminator, NPTII, CP4-EPSPS and endogenous gene Lectin. The research aims to build a method which is accurate, rapid and reliable for detection of genetically modified soybeans oil. The targeted gene including DNA was successfully established by the improved method, and then amplified by PCR. Five genes are simultaneously specifically detected. Commercial soybean, genetically modified soy bean and oil were detected with the Multiplex PCR. The improved method of DNA extraction was rapid and accurate to extract high quality total DNA which was amplified by PCR. The method could eliminate the PCR inhibitor. A way of detecting the genetically modified soybean and Oil was set up in this study.
文摘A PCR-ELISA method for detecting the glyphosate resistant transgenic soybean was established and optimized. The results showed that the key parameters of PCR-ELISA were as follows: the concentration of digoxin tag probe was 0.5 μmol · L^-1, the time of hybridization reaction was 15 min and the chromogenic reaction should last for 30 min. The sensitivity and the repeatability of our PCR-ELISA method were evaluated, and the results showed that it could be detected when the concentration of DNA template from transgenic soybean samples was 0.01% or higher, and the coefficient of variation of this method was less than 5% in our research condition. These results suggested that PCR-ELISA method establishment in this study had good repeatability and high precision for detecting the transgenic soybean samples.
基金Supported by Creative Research Groups of Heilongjiang Province of China(JC2016004)the National Key R&D Program of China(2016YFD0100201)Harbin Science Technology Project(2015RQXXJ018)。
文摘ENHANCER OF SHOOT REGENERATION(ESR1)is an important regulator of plant regeneration in vitro,which promotes regeneration of plant.In this study,transgenic positive plants with normal expression of proteins were screened by molecular assay.Through the study of the transgenic plants and the control Dongnong 50,the difference between immature embryo-induced callus and induced shoot bud was observed.The increase in callus weight indicated that GmESR1 gene accelerated the formations of shoot buds.By measuring the changes of hormone in the process of induction callus of transgenic plants,it was found that the contents of indole-3-acetic acid(IAA)and zeatin(ZT)in transgenic lines were significantly increased.It could be concluded that GmESR1 gene promoted the accumulation of hormone and affected regeneration process.In addition,this study also verified the interaction between GmBIM1 gene and GmESR1 gene by bimolecular fluorescence complementation(BiFC).
基金Supported by the Special Scientific Fund for Non-profit Environmental Industry(2010467038)
文摘Drought is a bottleneck for worldwide soybean production which is getting more serious as the climate continues to worsen. Dehydration responsive element binding(DREB) is a kind of transcription factor that regulates the expression of stress tolerance-related genes in response to drought, high salinity and cold stress in plant. Soybean with DREB gene possesses the drought resisting capability which is helpful to increase the yield. However, the potential risk of genetically modified plants(GMPs) on soil microbial community is still in debate. In order to understand the effects of transgenic DREB soybean on the nitrogen-fixing bacteria, the diversity of nif H gene in pot experiments planted transgenic soybean and near-isogenic nontransgenic soybean under normal water condition and drought stress condition was analyzed by PCR-DGGE and sequence analysis. The results showed that transgenic soybean under normal water condition decrease the diversity of the nitrogen-fixing bacteria in the seeding stage and flowering stage, but had no notable effect in other stages. Under drought stress, transgenic soybean reduced the diversity of the nitrogen-fixing bacteria in the flowering stage, but had no notable effects on other stages. Phylogenic analysis revealed that g7, g13, g15 and g19 had a close relationship with Alphaproteobacteria, g12 had a close relationship with Azonexus, others were related to Betaproteobacteria and Burkholderia.
基金Supported by National Science and Technology Major Project (2018ZX08020001-006)。
文摘[Objectives] This study was conducted to investigate the possibility of screening for herbicide resistance in the early stage of rapes growth. [Methods] Eight treatments were set for a herbicide concentration experiment. [Results] Rape sprouts were highly sensitive to the herbicide glyphosate, and even a very low concentration of glyphosate solution(187 mg/L) was sufficient to kill most of the rape seedlings, while the genetically modified herbicide-tolerant rape could tolerate higher concentrations of the herbicide. Low concentrations of glyphosate could be used for screening at the sprout stage, and the results of herbicide-tolerance screening were consistent with PCR testing. [Conclusions] This study lays a technical and material foundation for accelerating the cultivation of new herbicide-resistant rape varieties suitable for rapeseed production areas in the lower reaches of the Yangtze River.
基金supported by the Genetically Modified Organisms Breeding Major Projects of China (2016ZX08011-003)China Agriculture Research System (CARS-04)CAAS Agricultural Science and Technology Innovation Project
文摘Genetically modified(GM) organisms are widely adopted. However, their safety assessments and control are still of special concern to the public. Identifying and localizing transgene insertion is an essentially prerequisite step. In this study, 2 independent transgene soybean lines were selected(LB4-AtDCGS-1-20-5-2 and CGS-ZG11) as typical cases. Both lines contained expression cassette of At-DCGS that encoding a feedback-insensitive cystathionine gamma-synthase to produce higher level methionine(Met). LB4-AtDCGS-1-20-5-2 was whole genome sequenced with one paired-end 500 bp library and two mate-paired 1 kb and 2 kb libraries using Illumina HiSeq sequencing platform. CGS-ZG11 was sequenced with only one paired-end 500 bp library. Both genomes were assembled,and 2 scaffold sequences(1 for each line) were screened out by aligning with transgene.Then the transgene insertion and its flanking regions in soybean genome were further identified and confirmed by PCR cloning and Sanger sequencing. Results showed that these 2 transgene lines had single copy of inserted transgene. Their transgene insertion contents were identified, which facilitates further safety assessment. These results indicated that genome assembly using high throughput sequencing is a powerful tool for identifying transgene insertions, even with limited knowledge.
基金Supported by Important National Science & Technology Specific Pro-jects(2008ZX08012-001)~~
文摘[Objective] The aim was to establish an event-specific qualitative PCR method for transgenic soybean MON89788.[Method] Firstly,the 3′-junction sequence between host plant DNA and integrated DNA of transgenic MON89788 soybean was isolated using thermal asymmetric interlaced-PCR (TAIL-PCR),and the specific PCR primers were designed based on the 3′-junction sequence.Secondly,the specificity and sensitivity of the qualitative PCR detection methods employing these primers were tested.[Result] 1 142-bp 3′-junction sequence was obtained.According to the sequence,event-specific qualitative PCR method was established,amplifying a 170-bp product specifically from MON89788 event,and the limit of detection was 0.05%,approximately 40 initial template copies.[Conclusion] The method was highly specific,sensitive,and suitable for detection of MON89788 event.
基金financially supported by the National Transgenic Major Program, China (2016ZX08004001-04)
文摘Glyphosate is a highly efficient, broad-spectrum nonspecific herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)-mediated pathway of shikimic acid. The screening of glyphosate-resistant EPSPS gene is a major means for the development of new genetically modified glyphosate-resistant transgenic crop. Currently, the main commercialized glyphosate-resistant soybean contains glyphosate-resistant gene CP4-EPSPS. In this study, a G10-EPSPS gene was reported providing glyphosate resistance in Zhongdou 32. Here, G10-EPSPS gene was introduced into soybeans through Agrobacterium-mediated soybean cotyledon node. PCR, Southern blotting, semi-quantitative RT-PCR, qRT-PCR, and Western blotting were used, and the results revealed that G10-EPSPS had been integrated into the soybean genome and could be expressed steadily at both mRNA and protein levels. In addition, glyphosate resistance analysis showed that the growth of transgenic soybean had not been affected by concentrations of 900 and 2 700 g a.e. ha–1 of glyphosate. All the results indicated that G10-EPSPS could provide high glyphosate resistance in soybeans and be applied in production of glyphosate-resistant soybean.